Immunoglobulins and immunoglobulin fragments represent the most prevalent biopharmaceutical products in either manufacture or development worldwide. The high commercial demand for and hence value of this particular therapeutic market has led to the emphasis being placed on pharmaceutical companies to maximize the productivity of their respective manufacturing processes whilst controlling the associated costs.
Affinity chromatography, typically on matrices comprising staphylococcal Protein A or variants thereof, is normally used as one of the key steps in the purification of intact immunoglobulin molecules. The highly selective binding of Protein A to the Fc chain of immunoglobulins provides for a generic step with very high clearance of impurities and contaminants.
For antibody fragments, such as Fab, single-chain variable fragments (scFv), bi-specific T-cell engagers (BiTEs), domain antibodies etc., which lack the Fc chain but have a subclass 1,3 or 4 kappa light chain, matrices comprising Protein L derived from Peptostreptococcus magnus (B Åkerström, L Björck: J. Biol. Chem. 264, 19740-19746, 1989; W Kastern et al: J. Biol. Chem. 267, 12820-12825, 1992; B H K Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992 and U.S. Pat. No. 6,822,075) show great promise as a purification platform providing the high selectivity needed. The Protein L disclosed in U.S. Pat. No. 6,822,075 comprises the amino acid sequence SEQ ID NO: 1 plus an additional AVEN sequence at the N-terminus.
(Protein L)SEQ ID NO: 1KEETPETPETD SEEEVTIKAN LIFANGSTQT AEFKGTFEKA TSEAYAYADT LKKDNGEYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADA LKKDNGEYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFAEA TAEAYRYADL LAKENGKYTA DLEDGGYTIN IRFAGKKVDEKPEEProtein L matrices are commercially available as Capto™ L from GE Healthcare Bio-Sciences AB, Sweden (Capto L data file 29-0100-08 AC, 2014) and can be used for separation of kappa light chain-containing proteins such as intact antibodies, Fab fragments, scFv fragments, domain antibodies etc. About 75% of the antibodies produced by healthy humans have a kappa light chain and many therapeutic monoclonal antibodies and antibody fragments contain kappa light chains.
Any bioprocess chromatography application requires comprehensive attention to definite removal of contaminants. Such contaminants can for example be non-eluted molecules adsorbed to the stationary phase or matrix in a chromatographic procedure, such as non-desired biomolecules or microorganisms, including for example proteins, carbohydrates, lipids, bacteria and viruses. The removal of such contaminants from the matrix is usually performed after a first elution of the desired product in order to regenerate the matrix before subsequent use. Such removal usually involves a procedure known as cleaning-in-place (CIP), wherein agents capable of eluting contaminants from the stationary phase are used. One such class of agents often used with chromatography media is alkaline solutions that are passed over the matrix. At present the most extensively used cleaning and sanitizing agent is NaOH, and it is desirable to use it in concentrations ranging from 0.05 up to e.g. 1 M, depending on the degree and nature of contamination. Protein L is however a rather alkali-sensitive protein compared to e.g. Protein A and only tolerates up to about 15 mM NaOH over a large number of cycles. This means that additional, less desirable cleaning solutions, e.g. urea or guanidinium salts, may also have to be used in order to ensure sufficient cleaning.
An extensive research has earlier been focused on the development of engineered protein A ligands that exhibit an improved capacity to withstand alkaline pH-values. For example, WO2003/080655A1 discloses that Protein A domains with particular asparagine mutations are considerably more alkali stable than the native protein.
There is thus still a need in this field to obtain a separation matrix containing Protein L-derived ligands having an improved stability towards alkaline cleaning procedures.